Process of Evaluating Blood-Brain Barrier Permeability of Stroke Rat by Using Fluorescent Substance

ABSTRACT

A process of evaluating blood-brain barrier permeability of a stroke rat by using fluorescent substance is disclosed. This process uses an Evans blue dye having spontaneous fluorescence properties, in combination with the use of a new non-invasive in vivo imaging system (IVIS), to obtain fluorescent signals so as to assess the change in the blood-brain barrier permeability of rodents after a cerebral artery stroke model surgery. In operation, an Evans blue dye is injected into a stroke rat of middle cerebral artery occlusion model. A non-invasive in vivo imaging system is used to detect the fluorescence distribution of the whole brain, and obtain images combined by the fluorescence images and optical images for the whole brain tissue. Thereby, the change in the blood-brain barrier permeability of the stroke rat can be completely realized.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process of evaluating blood-brainbarrier permeability of a stroke rat by using fluorescent substance.Particularly, this invention relates to an evaluation process which canassess the change in the blood-brain barrier permeability of rodentsafter a cerebral artery stroke model surgery. More particularly thisinvention relates to an evaluation process which can prove the locationof the brain injury is fully consistent with the damaged region of theblood-brain barrier.

2. Description of Related Art

An Evans blue dye has high affinity to the albumin in the blood. Thealbumin cannot enter the brain through the blood-brain barrier. For thisreason, the Evans blue dye is often used to assess the blood-brainbarrier permeability. The Evans blue dye can be excited to emitfluorescence. Currently, the evaluation of the change in the blood-brainbarrier permeability of a single section of brain tissue is performed bymeans of fluorescence detection using a fluorescence microscope, whichcannot obtain the fluorescence imaging of the whole brain.

The conventional technology which is only capable of observing thefluorescence for the single section of brain tissue by viewing the brainslices through the fluorescent microscopy cannot provide the user withthe fluorescence imaging of the whole brain required in actual use.Therefore, there is a need of a novel process of evaluating theblood-brain barrier permeability of a stroke rat, which meets the user'sneed.

SUMMARY OF THE INVENTION

This present invention aims at overcoming the shortages of the prior artand providing a novel process which can detect the fluorescencedistribution over the whole brain and assess the change in theblood-brain barrier permeability of rodents after a cerebral arterystroke model surgery. Furthermore, this invention aims at providing aprocess of evaluating the blood-brain barrier permeability of a strokerat by using fluorescent substance.

In order to achieve the above and other objectives, the process ofevaluating the blood-brain barrier permeability of a stroke rat by usingfluorescent substance includes injecting a Evans blue dye into a strokerat of middle cerebral artery occlusion model; detecting thefluorescence distribution of the whole brain by using a non-invasive invivo imaging system; and obtaining images combined by the fluorescenceimages with optical images for the whole brain tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an evaluation process of the presentinvention.

FIG. 2 is an optical image of the whole brain tissue after the injectionof Evans blue dye according to the invention.

FIG. 3 is an integrated image combined the fluorescence images with theoptical images by using an IVIS 100 system according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The aforementioned illustrations and following detailed descriptions areexemplary for the purpose of further explaining the scope of the presentinvention. Other objectives and advantages related to the presentinvention will be illustrated in the subsequent descriptions andappended tables.

FIG. 1 is a schematic view of an evaluation process of the presentinvention. As shown, the invention is a process of evaluating theblood-brain barrier permeability of a stroke rat by using fluorescentsubstance. The process of the invention uses Evans blue dye havingspontaneous fluorescence properties, in combination with the use of anew non-invasive in vivo imaging system (IVIS), to obtain fluorescentsignals so as to assess the change in the blood-brain barrierpermeability of rodents after a cerebral artery stroke model surgery. Inoperation, an Evans blue dye 11 is injected into a stroke rat 1 ofmiddle cerebral artery occlusion model. A non-invasive in vivo imagingsystem 12 is used to detect the fluorescence distribution of the wholebrain, and obtain images combined by the fluorescence images and opticalimages for the whole brain tissue. Thereby, the change in theblood-brain barrier permeability of the stroke rat can be completelyrealized.

FIG. 2 is an optical image of the whole brain tissue after the injectionof Evans blue dye according to the invention. FIG. 3 is an integratedimage combined the fluorescence images with the optical images by usingan IVIS 100 system according to the invention. As shown, in a preferredembedment, the invention uses a SD (Sprague Dawley) rat as the strokeanimal model subject to transient bilateral common carotid artery andright middle cerebral artery ligation. After the rat is anesthetized,the surgery procedure is described roughly as follows. The skull of therat is opened by drilling in order to find the right middle cerebralartery. A 10-0 needle is used to take the right middle cerebral arteryand rotate it 90 degrees. It is ensured that the blood cannot flowsmoothly under the observation by a microscope. Then, both sides of thecommon carotid artery are subject to ligation for 90 minutes. After thestitches have been removed, wounds are sutured to allow the rat to berecovered.

Three groups are provided in the experiment, in which a sham group is arat whose skull is opened without the bilateral common carotid arteryand right middle cerebral artery ligation so as to exclude the impactcaused by drilling the skull; a control group is the blank rat withoutany surgery; and a surgery group is a stroke rat whose skull is openedto perform the middle cerebral artery ligation.

Evans blue dye is dissolved in saline and injected intravenously intothe rat via its tail 24 hours after surgery. After distribution for 30minutes, the rat is anesthetized for transcardial perfusion in order towash away the dye within the blood vessels. The brain tissue iscarefully taken and subject to digital camera photography (as shown inFIG. 2). The IVIS 100 (Caliper) system is used to detect thefluorescence distribution of the whole brain tissue. The Evans blue dyecan be excited by the light of 620 nanometer (nm) wavelengths to emitthe light of 680 nm wavelength which can be detected by the IVIS 100system and further converted into images (as shown in FIG. 3).

After the injection of Evans blue dye in intravenous way via the rat'stail 24 hours after stroke surgery, the dye will leak into theinterstitial cells of the right brain. As shown at the right side ofFIG. 2, the blue part represents the blue dye. The control group asshown at the left side of FIG. 2 and the sham group as shown at themiddle of FIG. 2 show no presence of the blue dye in the brain tissue.Furthermore, 24 hours of stroke after surgery, fluorescence signals aredetected in the right brain.

The location where emits the fluorescence can be found by integration ofthe optical images and the fluorescence images, which is consistent withthe location of the blue dye as shown at the right side of FIG. 3, andalso fully consistent with the part which is subject to the middlecerebral artery ligation. For the control group as shown at the leftside of FIG. 3 and the sham group as shown in the middle of FIG. 3,fluorescence is not obvious in the brain tissue.

Therefore, the present invention can prove that the location of thebrain injury is fully consistent with the damaged region of theblood-brain barrier by obtaining the clear image of the whole brain. Itis also confirmed that after the stroke, the blood-brain barrierpermeability deteriorates and allows the dye which originally cannotenter the brain to flow into the damaged brain area.

In this way, the present invention has successfully established a noveltechnology platform which can detect the relative position of thephotoluminescence of the whole brain. It is a convincing proof that thedye which should not exist at the location where the brain injury occursbut after stroke it can flow into that location. It means the strokewill destroy the blood-brain barrier permeability.

In summary, the process of evaluating the blood-brain barrierpermeability of a stroke rat by using fluorescent substance according tothe present invention effectively improves the shortcomings of the priorart. The Evans blue dye is injected into the rat of cerebral arteryocclusion model rat. The non-invasive in vivo imaging system (IVIS) isused to detect the fluorescence distribution over the whole brain.Thereby, the change in the blood-brain barrier permeability of thestroke rat can be completely understood.

The descriptions illustrated supra set forth simply the preferredembodiments of the present invention; however, the characteristics ofthe present invention are by no means restricted thereto. All changes,alternations, or modifications conveniently considered by those skilledin the art are deemed to be encompassed within the scope of the presentinvention delineated by the following claims.

The claims are as follows:
 1. A process of evaluating blood-brainbarrier permeability of a rat model of stroke by using a fluorescentsubstance, the process comprising: injecting an Evans blue dye into arat model of stroke, wherein the rat model of stroke has been subjectedto middle cerebral artery occlusion; detecting the fluorescencedistribution in the whole brain by using a non-invasive in vivo imagingsystem; and obtaining both fluorescence images and optical images forthe whole brain tissue.
 2. The process of claim 1, wherein the rat modelof stroke is subjected to transient bilateral common carotid artery andright middle cerebral artery ligation.
 3. The process of claim 1,wherein the fluorescence images and the optical images of the wholebrain tissue enable identification of where damage has occurred in thebrain.